The long term goal of this project is to better understand the genetic processes that regulate tooth development, to ultimately work toward gene therapy and tooth replacement. The project focuses on transcriptional regulation of the gene encoding EMSP1, which is a serine protease upregulated during the maturation stage of amelogenesis. The hypotheses are that EMSP1 is expressed in a highly restricted, tissue-specific manner, and that by defining the molecular signals involved in regulating EMSP1 expression, insight will be gained into the regulatory cascade causing the transition from secretory to maturation stages of amelogenesis. The three Specific Aims are: (1) To generate and characterize a beta-galactosidase knock-in mouse to precisely define the temporal and spatial expression of the EMSP1 gene; (2) to define the limits of the mouse EMSP1 promoter; and (3) to characterize the mouse EMSP1 promoter region. The knockin mouse will have a reporter gene transcribed from the endogenous EMSP1 promoter which will be used to determine the precise temporal and spatial expression of EMSP1. This pattern will be compared to that in transgenic mice and transfected cell lines that include cutback promoter-reporter constructs, in order to define the minimal promoter that gives normal expression.